Integrated Morphological, Comparative Transcriptomic, and Metabolomic Analyses Reveal Mechanisms Underlying Seasonal Patterns of Variation in Spines of the Giant Spiny Frog (Quasipaa spinosa).

Quasipaa spinosa, commonly known as the spiny frog, is an economically valued amphibian in China prized for its tender meat and nutritional value. This species exhibits marked sexual dimorphism, most notably the prominent spiny structures on males that are pivotal for mating success and species identification. The spines of Q. spinosa exhibit strong seasonal variation, changing significantly with the reproductive cycle, which typically spans from April to October. Sexually mature males develop densely packed, irregularly arranged round papillae with black spines on their chests during the breeding season, which may then reduce or disappear afterward, while females have smooth chest skin. Despite their ecological importance, the developmental mechanisms and biological functions of these spines have been inadequately explored. This study integrates morphological, transcriptomic, and metabolomic analyses to elucidate the mechanisms underlying the seasonal variation in spine characteristics of Q. spinosa. Our results demonstrate that spine density inversely correlates with body size and that spine development is accompanied by significant changes in epidermal thickness and keratinization during the breeding season. Comparative transcriptomic analysis across different breeding stages revealed significant gene expression alterations in pathways related to extracellular matrix interactions, tyrosine metabolism, Wnt signaling, and melanogenesis. Metabolomic analysis further identified significant seasonal shifts in metabolites essential for energy metabolism and melanin synthesis, including notable increases in citric acid and ß-alanine. These molecular changes are consistent with the observed morphological adaptations, suggesting a complex regulatory mechanism supporting spine development and functionality. This study provides novel insights into the molecular basis of spine morphogenesis and its seasonal dynamics in Q. spinosa, contributing valuable information for the species' conservation and aquaculture.

Comparison of Lipid Composition between Quasipaa spinosa Oil and Rana catesbeiana Oil and Its Effect on Lipid Accumulation in Caenorhabditis elegans.

Frog oil has been recognized for its nutritional and medicinal value. However, there is limited research on the role of frog oil in preventing obesity. In this study, we aimed to investigate the lipid composition of Quasipaa spinosa oil (QSO) and Rana catesbeiana oil (RCO) using lipidomics analysis. We compared the lipid accumulation effects of these two kinds of frog oils and soybean oil (SO) in Caenorhabditis elegans (C. elegans). Additionally, we determined the gene expression related to lipid metabolism and used the nhr-49 mutant (RB1716) and sir-2.1 mutant (VC199) for validation experiments. The results showed that the lipid composition of QSO and RCO was significantly different (p < 0.05), and QSO was rich in more polyunsaturated fatty acids (PUFAs). After feeding C. elegans, the lipid accumulation of the QSO group was the lowest among the three dietary oil groups. In addition, compared with RCO and SO, QSO significantly inhibited the production of malondialdehyde (MDA) and increased the activity of superoxide dismutase (SOD). The effects of three kinds of dietary oils on the fatty acid composition of C. elegans were significantly different. Compared with SO and RCO, QSO significantly up-regulated (p < 0.05) the expression of sir-2.1 and ech-1 genes. The results showed that QSO might reduce lipid accumulation through the SIRT1 and nuclear hormone signaling pathways. Such a situation was verified experimentally by the nhr-49 mutant (RB1716) and sir-2.1 mutant (VC199). This study proposed a new functional oil, laying the groundwork for developing functional foods from Quasipaa spinosa.

Identification and drug resistance of pathogen of ulcerative skin disease and its immune responses and protective efficacy after vaccination in a giant spiny frog, Quasipaa spinosa.

OBJECTIVE: In order to explore the pathogen of the ulcerative skin disease in giant spiny frog (Quasipaa spinosa), and to provide theoretical basis for the prevention and control of the disease in practical production, this study was carried out to isolate and identify the pathogenic bacteria from the sick frogs suffering from rotting skin disease and to carry out the immunization test of the inactivated vaccine. METHODS: Physiological and biochemical characterization, and molecular biology of the pathogenic bacteria were identified, and drug screening and immunization responses were also carried out. RESULTS: The dominant bacterium QS01 was isolated from the lesions of diseased giant spiny frogs, which was confirmed to be the causative agent of the rotting skin disease of giant spiny frogs by artificial regression infection test. Based on the fact that the pathogen is a gram-negative short bacterium, its phenotypic characteristics and 16S rRNA and gyrB gene sequences were analyzed, and the bacterium was determined to be Citrobacter freundii. The results of the drug sensitivity test showed that the bacterium was sensitive to 11 antibiotics, including Enrofloxacin, Fleroxacin and Ciprofloxacin, including three non-polluting drugs such as Florfenicol, Roxithromycin and Thiamphenicol, as well as three Chinese herbal medicines such as Rheum officinale Baill, Coptis chinensis Franch and Scutellaria baicalensis Georgi. Most non-specific immune responses could go to recovery in 24h. The frogs were vaccinated with QS01 formaldehyde inactivated vaccine by injection, immersion and spraying, and the serum antibody potency of the three immunized groups with the average potency reached the peak at the 20th d after immunization, and the serum antibody potency of the injected immunized group was at the highest ratio of 1:64-128 (101.6), while the immersed group and the spraying group attained the ratio of 1:16-32 (20.2) and 1:16-32 (16) respectively, and lasted until the 30th d. The control group that was not immunized had the highest serum antibody potency of 1:16-32 (20.2) and 1:16-32 (16), and continued until the 30th d. The control group that was not immunized was not immunized. The serum antibody potency of the unimmunized control group was 1:2 to 2(2). The immunoprotection rates after takedown were 100 %, 85.71 % and 71.43 %, respectively. CONCLUSION: C. freundii is the pathogen of the disease in this farm, and the vaccination by immersion and spraying can effectively prevent and control the rotting skin disease in frogs. These results revealed pathogenicity of C. freundii and its activation of host immune response, which will provide a scientific reference for the aquaculture and disease prevention in Q. spinosa culture.

Transcriptomic analysis of skin immunity genes in the Chinese spiny frog (Quasipaa spinosa) after Proteus mirabilis infection.

Recently, populations of Chinese spiny frogs (Quasipaa spinosa), an important amphibian species in China, have decreased, mainly due to a disease caused by the gram-negative bacteria Proteus mirabilis. To elucidate the immune response of the frogs, this study aimed to identify novel candidate genes functionally associated with P. mirabilis infection-induced "rotting skin" disease. Chinese spiny frogs were infected with P. mirabilis, and the skin transcriptome was sequenced using the MGISEQ-2000 platform. A total of 233,965 unigenes were obtained by sequencing, of which 27.23 % were known genes. Screening of differentially expressed genes (DEGs) indicated 210 unigenes differentially expressed after P. mirabilis infection, of which 132 unigenes were up-regulated, and 78 unigenes were down-regulated. Using Kyoto Encyclopedia of Genes and Genomes enrichment analysis, DEGs were identified as enriched in signal pathways, such as oxidative phosphorylation, apoptosis, and the Janus kinase-signal transducer and activator of transcription pathway. Of the DEGs, there was a significant upregulation of the colony stimulating factor 2 receptor beta common subunit, interleukin 2 receptor subunit gamma, cathelicidin antimicrobial peptide, interleukin-17 receptor E, receptor-interacting serine/threonine-protein kinase 3, and pulmonary surfactant-associated protein D immune genes following P. mirabilis infection. Conversely, scavenger receptor cysteine-rich domain-containing group B protein, tumor protein p53 inducible nuclear protein 2, suppressor of cytokine signaling 2, and metalloreductase STEAP3 were significantly downregulated. In conclusion, the first skin transcriptome database of Chinese spiny frogs was established, and several immune genes were identified to elucidate the pathogenic mechanism of "skin rot" in Chinese spiny frogs and other cultured frogs.

Prediction of Potential Suitable Distribution Areas of Quasipaa spinosa in China Based on MaxEnt Optimization Model.

Quasipaa spinosa is a large cold-water frog unique to China, with great ecological and economic value. In recent years, due to the impact of human activities on the climate, its habitat has been destroyed, resulting in a sharp decline in natural population resources. Based on the existing distribution records of Q. spinosa, this study uses the optimized MaxEnt model and ArcGis 10.2 software to screen out 10 factors such as climate and altitude to predict its future potential distribution area because of climate change. The results show that when the parameters are FC = LQHP and RM = 3, the MaxEnt model is optimal and AUC values are greater than 0.95. The precipitation of the driest month (bio14), temperature seasonality (bio4), elevation (ele), isothermality (bio3), and the minimum temperature of coldest month (bio6) were the main environmental factors affecting the potential range of the Q. spinosa. At present, high-suitability areas are mainly in the Hunan, Fujian, Jiangxi, Chongqing, Guizhou, Anhui, and Sichuan provinces of China. In the future, the potential distribution area of Q. spinosa may gradually extend to the northwest and north. The low-concentration emissions scenario in the future can increase the area of suitable habitat for Q. spinosa and slow down the reduction in the amount of high-suitability areas to a certain extent. In conclusion, the habitat of Q. spinosa is mainly distributed in southern China. Because of global climate change, the high-altitude mountainous areas in southern China with abundant water resources may be the main potential habitat area of Q. spinosa. Predicting the changes in the distribution patterns of Q. spinosa can better help us understand the biogeography of Q. spinosa and develop conservation strategies to minimize the impacts of climate change.

Transcriptome characterization and SSR discovery in the giant spiny frog Quasipaa spinosa.

The giant spiny frog Quasipaa spinosa (Amphibia: Ranidae) is a large unique frog species found mainly in southern China with a low amount of fat and high protein, and it has become one of the most important aquaculture animal species in China. To better understand its genetic background and screen potential molecular markers for artificial breeding and species conservation, we constructed an expression profile of Q. spinosa with high-throughput RNA sequencing and acquired potential SSR markers. Approximately 81.7 Gb of data and 93,887 unigenes were generated. The transcriptome contains 2085 (80.7 %) complete BUSCOs, suggesting that our assembly methods were effective and accurate.These unigenes were functionally classified using 7 functional databases, yielding 17,482 Pfam-, 12,752 Sting-, 17,526 KEGG-, 24,341 Swiss-Prot-, 28,604 Nr-, 16,287 GO- and 12,752 COG-annotated unigenes. Among several amphibian species, Q. spinosa unigenes had the highest number of hits to Xenopus tropicalis (35.25 %), followed by Xenopus laevis (12.68 %). 1417 unigenes were assigned to the immune system. In addition, a total of 33,019 candidate SSR markers were identified from the constructed library. Further tests with 20 loci and 118 large-scale breeding specimens gathered from four culture farms in China showed that 15 (75 %) loci were polymorphic, with the number of alleles per locus varying from 3 to 9 (mean of 4.3). The PIC values for the SSR markers ranged from 0.19 to 0.82, with an average value of 0.43, indicating moderate polymorphism in Q. spinosa. The transcriptomic profile and SSR repertoire obtained in the present study will facilitate population genetic studies and the selective breeding of amphibian species.

Ontogenetic characteristics of the intestinal microbiota of Quasipaa spinosa revealed by 16S rRNA gene sequencing.

To analyse the correlation between the intestinal microbiota (IM) and differential development of Quasipaa spinosa across different stages of metamorphosis, we focused on four key developmental periods (15 days post-hatch [dph; Gosner stage, GS23], 60 dph [GS25], 90 dph [GS41] and 150 dph [GS25, GS41, GS42, GS46]) and used 16S rDNA amplicon sequencing. Results showed that IM diversity in Q. spinosa was related to life history. Specifically, there was a significant difference between the aquatic and terrestrial stages, and IM diversity increased with age. Proteobacteria, Bacteroidetes and Firmicutes were the dominant phyla in tadpoles, whereas Bacteroidetes, Proteobacteria, Firmicutes and Fusobacteria were the dominant phyla at the metamorphosis stages. The composition of the IM at different stages and ages varied considerably, but the changes were not substantial among different development stages at 150 dph. This was probably because age has a greater influence than growth factors, and host selective pressure increases with age. This study provides a theoretical reference for subsequent studies on the IM, as well as for the prevention of amphibian endogenous pathogenic microbial diseases.

A chromosomal level genome sequence for Quasipaa spinosa (Dicroglossidae) reveals chromosomal evolution and population diversity.

Quasipaa spinosa is an Asian commercial Dicroglossidae species noted for its spiny chest found in adult males. Here, we report the first chromosomal level Q. spinosa genome employing PacBio long read sequencing and high-resolution chromosome conformation capture (Hi-C) technology. The total length of the final assembled genome was 2,839,292,578 bp, with contig N50 of 3.79 Mb and scaffold N50 of 327.44 Mb. Approximately 99.30% of the length of the assembled genome sequences were anchored to 13 chromosomes with the assistance of Hi-C reads. A total of 26,173 protein-coding genes were predicted, and 95.98% of the genes were functionally annotated. The annotated genes covered a total of 92.10% of the complete vertebrate core gene set according to the BUSCO pipeline evaluation. Approximately 41 million years ago, Q. spinosa began to diverge from its dicroglossid sister taxon Nanorana parkeri. The Q. spinosa genome revealed obvious chromosomal fissions compared with Xenopus tropicalis, which probably represented a specific chromosome evolutionary history within frogs. Population analysis showed that Chinese Q. spinosa could be divided into eastern and western genetic clusters, with the western population showing higher diversity than the eastern population. The effective population size of Q. spinosa showed a continuously decreasing trend from one million years ago to 10,000 years ago. In summary, this study sheds light on Q. spinosa evolution and population differentiation, providing a valuable genomic resource for further biological and genetic studies on this species, and other closely related frog taxa.

Functional differences of three CXCL10 homologues in the giant spiny frog Quasipaa spinosa.

Chemokines are a superfamily of structurally related chemotactic cytokines exerting significant roles in acting as a bridge between the innate and adaptive immune responses. In this study, we identified three CXC motif chemokine 10 (CXCL10) homologues (QsCXCL10-1, QsCXCL10-2 and QsCXCL10-3) from giant spiny frog Quasipaa spinosa. All three deduced QsCXCL10 proteins contained four conserved cysteine residues as found in other known CXC chemokines. Phylogenetic analysis showed that QsCXCL10-1, 2, 3 and other CXCL10s in amphibian were grouped together to form a separate clade. These three QsCXCL10s were highly expressed in spleen and blood. Upon infection with Staphylococcus aureus or Aeromonas hydrophila, the expressions of QsCXCL10s were markedly increased in spleen and blood during biotic stresses. Meanwhile, the QsCXCL10s transcription in liver could also be up-regulated under abiotic stresses such as cold and heat stresses. The recombinant proteins of frog CXCL10 homologues were produced and purified in E. coli and possessed similar but differential bioactivities. Both rCXCL10-1 and rCXCL10-2 had strong effects on the up-regulation of pro-inflammatory cytokines (TNF-α, IL-1ß and IL-8) in vivo, whereas rCXCL10-3 induced a weak expression of these cytokines. Moreover, the rCXCL10-1 and rCXCL10-2 could strongly promote splenocyte proliferation and induce lymphocytes migration, while rCXCL10-3 had limited effects on these biological processes. All three frog chemokines triggered their functional activities by engaging CXC motif chemokine receptor 3 (CXCR3). Taken together, these results revealed that the three QsCXCL10s had similar but differential functional activities in mediating immune responses and host defenses, which might contribute to a better understanding of the functional evolution of CXCL10 in vertebrates.

Elizabethkingia miricola infection in Chinese spiny frog (Quasipaa spinosa).

Elizabethkingia miricola is a Gram-negative rod which has been incriminated in severe infections in humans. Recently, a serious infectious disease was identified in Chinese spiny frogs (Quasipaa spinosa), in the Sichuan Province of China; the disease was characterized by corneal opacity, the presence of ascites and neurological symptoms. A Gram-negative bacillus was isolated from the liver, spleen and kidney of the diseased frogs. Experimental infection test revealed that the bacillus could infect the frogs Q. spinosa and the LD50 value was 1.19 × 106  cfu per frog. The isolated Gram-negative bacillus was identified as E. miricola according to phenotypic characteristics, 16S rRNA and gyrB gene sequence analysis. The isolated strain was only susceptible to florfenicol among all investigated chemotherapeutic agents. Histological examination revealed that E. miricola infection caused pathological lesions to multiple organs and tissues, especially in the liver, brain, kidney. These results confirmed that E. miricola is an emerging pathogen of Chinese spiny frogs.

Genetic polymorphism of major histocompatibility complex class IIB alleles and pathogen resistance in the giant spiny frog Quasipaa spinosa.

Major histocompatibility complex (MHC) genes are candidates for determining disease susceptibility due to their pivotal role in both innate and adaptive immune responses. Accordingly, the association between the genetic variation of MHC genes and the pathogen resistance has been investigated in numerous vertebrates. To date, however, little is reported in amphibians. In this study, we investigate the genetic variation at the MHC class IIB gene in the giant spiny frog Quasipaa spinosa, which has high commercial value in China. The full length of MHC class IIB cDNA was cloned from Q. spinosa by homology cloning and rapid amplification of cDNA end-polymerase chain reaction (RACE-PCR). Two MHC class IIB loci were identified in Q. spinosa. We also developed PCR primers for a portion of the second exon of the MHC class IIB gene. A total of 26 MHC class IIB alleles were identified. The dN rate was significantly higher than the dS rate in the putative peptide-binding region, thereby proving the positive selection hypothesis. In addition, individuals intraperitoneally injected with Aeromonas hydrophila were used to study the association between MHC class IIB alleles and pathogen resistance/susceptibility, to explore the specific alleles in balancing selection. Eighty frogs were used after exposure to A. hydrophila infection. Nine alleles were used to study the association between the alleles and disease resistance. Two alleles, namely, Pasa-DAB(∗)1301 and Pasa-DAB(∗)0901, were significantly associated with resistance against A. hydrophila. This study provides valuable information on the structure of the MHC class IIB gene and confirms the association between MHC class IIB gene alleles and disease resistance to bacterial infection in Q. spinosa. Moreover, pathogen resistance-related MHC markers can be used for the selective breeding of the giant spiny frog.